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Tumor-Infiltrating Lymphocytes (TIL), Tertiary Lymphoid Structures (TLS), and Expression of PD-1, TIM-3, LAG-3 on TIL in Invasive and In Situ Ductal Breast Carcinomas and Their Relationship with Prognostic Factors

Published:August 19, 2022DOI:https://doi.org/10.1016/j.clbc.2022.08.005

      Abstract

      Introduction

      Immunotherapy has been determined as an important choice in breast carcinomas, especially in tumors with markedly inflammatory response. About this promising subject, tumor-infiltrating lymphocytes (TIL) and the expression of immune control point receptors on TIL have gained importance.

      Materials and Methods

      In this study, stromal TIL and tertiary lymphoid structures (TLS) were determined in tumor tissues of 312 invasive and 68 in situ breast cancer patients. Expression rates of PD-1, LAG-3, and TIM-3 on intratumoral and stromal TIL were immunohistochemically evaluated.

      Results

      In invasive breast carcinomas, stromal TIL was found to be significantly associated with lymph node metastasis, HR and HER2 expression, and basal-like phenotype, as the presence of TLS with neoadjuvant therapy, recurrence, death, and expression of HR and HER2. PD-1, LAG-3, and TIM-3 expressions were found to be associated with HR and HER2 status, stromal TIL rates, and TLS. In multivariate analysis, high stromal TIL and PD-1 expression in intratumoral TIL were found to be independent prognostic factors in terms of overall survival and disease-free survival.

      Conclusion

      Evaluation of TIL and immune control point receptor expressions in breast cancer is particularly important in terms of planning the therapeutic approaches based on immunotherapy protocols.

      Keywords

      Abbreviations:

      TIL (Tumor-Infiltrating Lymphocytes), TLS (Tertiary Lymphoid Structures)

      Introduction

      Breast cancer is the most common cancer seen in women. The prevalence of breast cancer is increasing all over the world, and despite all kinds of treatment methods, it is reported that this cancer causes an average of 627,000 deaths per year.
      • Bray F
      • Ferlay J
      • Soerjomataram I
      • Siegel RL
      • Torre LA
      • Jemal A.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      In addition to surgical treatment, the treatment of breast cancer is shaped according to the hormone receptor status [HR (estrogen and progesterone receptors-ER and PR)], c-erbB-2 (HER2) expression, and Ki67 proliferation index of the tumor. As it is known, ER modulators such as tamoxifen are administered in patients with positive HR; monoclonal antibodies developed against the HER2 oncogene, such as trastuzumab, in patients with score 3 positive c-erbB-2; and, cytotoxic chemotherapeutic agents (such as paclitaxel and doxorubicin) in patients with negative HR and HER2 [triple (-)] tumors. These treatments shaped by the immunohistochemical staining profile may not always produce the desired results due to the molecular subtype difference of the tumor, additional diseases of the patient, and the side effects of the drugs used.
      • Prat A
      • Pineda E
      • Adamo B
      • et al.
      Clinical implications of the intrinsic molecular subtypes of breast cancer.
      However, in recent years, immunotherapy has become a promising treatment option for especially triple (-) breast carcinoma, accompanied by an inflammatory response. The studies have been started on the use of immunotherapeutic agents in combination or as a single agent in order to avoid the negative effects of chemotherapy and increase the effectiveness of treatment, especially in triple (-) breast cancer patients.
      • Brignone C
      • Gutierrez M
      • Mefti F
      • et al.
      First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity.

      Emens LA, Braiteh FS, Cassier P, et al. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer (TNBC). AACR; 2015. doi:10.1158/1538-7445.AM2015-2859

      • Migali C
      • Milano M
      • Trapani D
      • et al.
      Strategies to modulate the immune system in breast cancer: checkpoint inhibitors and beyond.
      Immunotherapy have been studied by oncologists and immunologists for many years. The effects of chemotherapy and radiotherapy on the death of proliferating cells and arrest of cell division affect not only tumor cells but also normal cells. This situation causes an increase in mortality and morbidity in breast cancer patients. Therefore, research and development of more specific treatment methods such as immunotherapy have gained speed.
      • Abbas AK
      • Lichtman AH
      • Pillai S.
      Cellular and Molecular Immunology E-Book.
      The most recent targets in immunotherapy are the immune control point receptors, which are programmed cell death protein-1 (PD-1) and its ligand (PD-L1), T-lymphocyte associated antigen-4 (CTLA-4), lymphocyte activation gene (LAG-3), T-cell immunoglobulin, and mucin domain-containing molecule 3 (TIM-3). These receptors are highly expressed in regulatory T-cells and have a negative role in T-cell proliferation and activation.
      • Anderson AC
      • Joller N
      • Kuchroo VK.
      Lag-3, Tim-3, and TIGIT: Co-inhibitory receptors with specialized functions in immune regulation.
      The efficacy of immune checkpoint molecules is being investigated in tumors involving immune cell infiltration. The first parameter to be determined is the density of tumor-infiltrating lymphocytes (TIL) within the tumor, and then, the expression of inhibitor receptors on these lymphocytes. It has been understood that these receptors, which are used as targets in treatment, are closely related to prognosis. It has been reported that the density of TIL in breast cancer is found to be related to both the neoadjuvant chemotherapy response and the prognosis of patients who will receive adjuvant chemotherapy. Also, TIL are thought to be effective in transforming an in situ tumor into an invasive tumor.
      • Dieci MV
      • Radosevic-Robin N
      • Fineberg S
      • et al.
      Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the international immuno-oncology biomarker working group on breast cancer.
      ,
      • Stanton SE
      • Disis ML.
      Clinical significance of tumor-infiltrating lymphocytes in breast cancer.
      Another subject that has recently been investigated within an invasive tumor is the relationship between tertiary lymphoid structures (TLS) with prognosis. TLS are ectopic lymphoid formations similar to secondary lymphoid organs.
      • Hiraoka N
      • Ino Y
      Yamazaki-Itoh R. tertiary lymphoid organs in cancer tissues.
      Previously, the presence of TLS in different cancers (lung cancer, colon cancer, and melanoma) has been investigated in terms of prognosis.
      • Dieu-Nosjean MC
      • Antoine M
      • Danel C
      • et al.
      Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures.
      • Trajkovski G
      • Ognjenovic L
      • Karadzov Z
      • et al.
      Tertiary lymphoid structures in colorectal cancers and their prognostic value.
      • Cabrita R
      • Lauss M
      • Sanna A
      • et al.
      Tertiary lymphoid structures improve immunotherapy and survival in melanoma.
      Studies also report that these structures have a prognostic significance in breast cancer.
      • Liu X
      • Tsang JYS
      • Hlaing T
      • et al.
      Distinct tertiary lymphoid structure associations and their prognostic relevance in HER2 positive and negative breast cancers.
      ,
      • Sofopoulos M
      • Fortis SP
      • Vaxevanis CK
      • et al.
      The prognostic significance of peritumoral tertiary lymphoid structures in breast cancer.
      In this study on invasive and in situ ductal breast carcinomas, we aimed to determine the relationship between the density of TIL with ER, PR, and HER2 expressions of tumor cells as well as the expression profiles of new generation immune checkpoint molecules (PD-1, LAG-3, and TIM-3) and to associate the obtained data with prognostic factors and survival of breast cancer patients.

      Materials and Methods

      Patient Selection

      A total of 380 breast cancer cases were included in this study whom 312 were diagnosed as invasive ductal carcinoma between the years 2010 to 2018 and 68 as ductal carcinoma in situ (DCIS) between 2010 and 2015 in the Department of Pathology, Faculty of Medicine, Gazi University, Ankara, TURKEY. The pathological materials consisting of hematoxylin and eosin (H&E) as well as immunohistochemically stained slides representing the tumor area obtained from total or partial mastectomy specimens were reassessed by 2 pathologists together.
      The pathology reports and medical files of the selected cases were screened for histopathological and clinical data, respectively. Patients with the diagnosis of invasive ductal carcinoma including those with medullary pattern were grouped according to their histological grades and ER-PR-HER2 expression status. Patients with DCIS were selected among those without any accompanying invasive carcinoma component (except microinvasion) and those previously diagnosed as invasive carcinoma via excisional biopsy or mastectomy was excluded from the study. The information including prognostic factors such as tumor size, histological grade, ER-PR-HER2 expression, Ki67 proliferation index, and metastatic lymph node status of the selected cases were obtained from pathology reports; whereas distant metastasis status, pathological stage, neoadjuvant treatment, recurrence, disease-free survival, and overall survival status from medical files via hospital information management system.
      The study was approved by the Gazi University Faculty of Medicine Clinical Research Ethics Committee in terms of ethics on April 29, 2019, with the meeting number 04.

      Histopathological Evaluation

      TIL evaluation was done retrospectively on H&E slides via light microscopy. While evaluating TIL, the guides of International Immuno-Oncology Biomarker Working Group on Breast Cancer and International TILs Working Group were considered.
      • Dieci MV
      • Radosevic-Robin N
      • Fineberg S
      • et al.
      Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the international immuno-oncology biomarker working group on breast cancer.
      ,
      • Salgado R
      • Denkert C
      • Demaria S
      • et al.
      The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.
      TIL can be evaluated separately in the stromal, intratumoral and, peritumoral areas in breast cancer. Stromal TIL is defined as the inflammatory cells among tumor groups, while intratumoral TIL as the inflammatory cells within the tumor islands. However, the evaluation of TIL in the stromal areas is recommended due to its repeatability and ease.
      • Salgado R
      • Denkert C
      • Demaria S
      • et al.
      The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.
      Therefore, in our study, we preferred evaluating stromal TIL.
      For the evaluation of TIL in invasive carcinoma cases, the best tissue samples representing the tumor were chosen according to the presence of stromal TIL density, lymphoid aggregates/TLS, and peritumoral parenchymal inflammation. Besides these features, presence of accompanying DCIS, with or without TIL, was noted. While making this choice, the quality of tissue processing related to the tumor area, which was considered to be very important, was also taken into consideration. The necrotic areas and areas with dense central hyalinization within the tumor were not evaluated. In tumor samples having a heterogeneous TIL density, whole-mount sections were examined at 200x magnification and the mean values were calculated.
      • Dieci MV
      • Radosevic-Robin N
      • Fineberg S
      • et al.
      Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the international immuno-oncology biomarker working group on breast cancer.
      ,
      • Salgado R
      • Denkert C
      • Demaria S
      • et al.
      The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.
      On the other hand, for the evaluation of DCIS, not only the density of stromal TIL was determined but morphological patterns (ie, micropapillary, cribriform, solid, and papillary), presence of comedonecrosis, apoptosis, calcification, and healing phenomenon were noted, as well.
      The density of stromal TIL is classified as mild (1%-10%), moderate (11%-49%), and high (≥ 50%) both in invasive and in situ ductal carcinomas.

      Immunohistochemical Evaluation and Scoring

      Cases with clinical follow-up information and density of stromal TIL of more than 3% were included in the immunohistochemical study (98 of invasive, 23 of in situ ductal carcinoma cases). The tumor samples were all formalin-fixed and paraffin-embedded tissues and approximately 4-micron thick sections were prepared from each tissue block for immunohistochemical examination. CD3, PD-1, LAG-3, and TIM-3 were stained immunohistochemically by using a closed automated slide stainer (Ventana Benchmark XT). Tonsil tissue was used as a positive control for all antibodies. The clones and concentrations of these antibodies were as follows: CD3 (clone 2GV6, ready to use), PD-1 (clone NAT105, ready to use), LAG-3 (clone D2G40, dilution 1:200) and TIM-3 (clone D5D5R, dilution 1:400).
      The density of intratumoral TIL was evaluated by determining the percentage of the tumor area occupied by CD3 positive T lymphocytes within tumor islands. The density of intratumoral TIL is classified similar to stromal TIL evaluation (ie, mild, medium, and high).
      Cytoplasmic and/or membranous PD-1, LAG-3 and TIM-3 staining in lymphocytes were considered specific for the immune reaction pattern. Stained lymphocytes were scored separately for intratumoral and stromal TIL. The ratio of stained lymphocytes to total lymphocytes was evaluated by examining 3 independent microscopic high-power fields (x400) and then expressed as a percentage value. According to this, less than 5% was considered as low expression, while 5% as high. Total lymphocyte count in the intratumoral area was determined by CD3 staining.

      Statistical Analyses

      The analysis of the data was done using IBM SPSS version 22. The relationship between TIL, TLS and, immune checkpoint molecules expression on TIL and clinical features was evaluated by chi-square test. The mean ± standard deviation for age was calculated and the mean age difference among the groups was evaluated with the One-Way Anova test. Kaplan-Meier method was used for the effect of clinicopathological characteristics of patients on survival and compared with the log-rank test. Univariate and multivariate Cox regression models were used to investigate independent prognostic factors. In all tests, P ≤ .05 was considered statistically significant.

      Results

      Baseline Clinical Features

      In our study, 312 invasive breast carcinoma cases aged between 26 and 84 years were reevaluated and further studied. The mean age was 52.2. Only 5 cases were male and the rest were female. The mean tumor diameter was 3.1 ± 2.3 cm. The median follow-up period of the cases was 68 months (min. 2 months, max. 123 months). The 5-year overall survival rate of these cases was found to be 80%, whereas the 10-year overall survival rate was 72%. We are able to access clinical follow-up information 169 out of 312 cases. With the data obtained, the median disease-free survival time was found to be 61 months (min. 6 months, max. 120 months). In patients with available clinical follow-up information, the 5-year and the 10-year disease-free survival rates were 72% and, 69%, respectively.
      In terms of DCIS, the number of cases was 68. The cases were aged between 29 and 77, with a mean of 53.8. All cases were female. The mean tumor diameter was 2.1 ± 2.5 cm.

      Stromal TIL Density: Relationship With Pathological Features and Prognosis

      High stromal TIL density was significantly found to be associated with basal-like phenotype and medullary-like invasive ductal carcinomas as expected (P < .001). It was also observed in triple-negative breast carcinomas and HR (-)/HER2 (+) tumors (P = .001); and also in ER and PR negative tumors (P < .001 and P = .004, respectively). Among other histological parameters examined, the relationship between TIL density of the invasive tumor with TLS and with accompanying DCIS is also found to be significant (P < .001) (Table 1). It was also shown that, in terms of DCIS, those cases who were HR (-)/HER2 (+) and those with tumor grade 3 of 3 had higher stromal TIL density (P < .001 and P = .022, respectively). In DCIS, as in invasive carcinomas, TLS were also found to be associated with higher stromal TIL density (P < .001). This was particularly noticeable in DCIS that show a healing phenomenon (P < .001) (Supplementary Table 1).
      Table 1The Relationship Between sTIL Density With Clinicopathological and Histological Parameters in Invasive Breast Tumors
      ParametersPatientsDensity of sTIL
      n (%)Mild (1%-10%)Moderate (11%-49%)High (≥50%)P
      Age at diagnosis3121767066.919
      Mean ± SD52.2 ± 12.852.5 ± 13.451.7 ± 11.552.1 ± 12.6
      Death312.084
      Present69 (22.1)47 (68.1)11 (15.9)11 (15.9)
      Absent243 (77.9)129 (53.1)59 (24.3)55 (22.6)
      Recurrence169.052
      Present46 (27.2)34 (73.9)8 (17.4)4 (8.7)
      Absent123 (72.8)67 (54.5)29 (23.6)27 (22.0)
      Neoadjuvant therapy169.029
      Present35 (20.7)25(71.4)9 (25.7)1 (2.9)
      Absent134 (79.3)76 (56.7)28 (20.9)30 (22.4)
      pT300.715
      pT199 (33.0)51 (51.5)25 (25.3)23 (23.2)
      pT2163 (54.3)96 (58.9)34 (20.9)33 (20.2)
      pT332 (10.7)19 (59.4)8 (25.0)5 (15.6)
      pT46 (2.0)4 (66.7)0 (-)2 (33.3)
      pN306.040
      pN0134 (43.8)80 (59.7)21 (15.7)33 (24.6)
      pN1-2-3172 (56.2)93 (54.1)47 (27.3)32 (18.6)
      pM305.272
      pM0298 (97.7)166 (55.7)68 (22.8)64 (21.5)
      pM17 (2.3)6 (85.7)0 (-)1 (14.3)
      TNM stages303.356
      I63 (20.8)32 (50.8)14 (22.2)17 (27.0)
      II138 (45.5)85 (61.6)27 (19.6)26 (18.8)
      III95 (31.4)48 (50.5)26 (27.4)21 (22.1)
      IV7 (2.3)6 (85.7)0 (-)1 (14.3)
      Histologic type312<.001
      Invasive ductal carcinoma, NOS263 (84.3)155 (58.9)64 (24.3)44 (16.7)
      Invasive ductal carcinoma with basal-like phenotype38 (12.2)19 (50)6 (15.8)13 (34.2)
      Invasive ductal carcinoma with medullary pattern8 (2.6)0 (-)0 (-)8 (100)
      Invasive ductal carcinoma showing signet ring cell morphology2 (0.6)2 (100)0 (-)0 (-)
      Invasive ductal carcinoma with metaplastic features1 (0.3)0 (-)0 (-)1 (100)
      Receptor expression307.001
      Triple (-)97 (31.6)48 (49.5)22 (22.7)27 (27.8)
      HR (-)/HER2 (+)64 (20.8)27 (42.2)15 (23.4)22 (34.4)
      HR (+)/HER2 (+)74 (24.1)51 (68.9)17 (23)6 (8.1)
      HR (+)/HER2 (-)72 (23.5)49 (68.1)13 (18.1)10 (13.9)
      Histologic grade312.676
      13 (1.0)3 (100)0 (-)0 (-)
      218 (5.8)11 (61.1)5 (27.8)2 (11.1)
      3291 (93.3)162 (55.7)65 (22.3)64 (22.0)
      ER expression307<.001
      Negative (0)157 (51.1)73 (46.5)36 (22.9)48 (30.6)
      Low (1%-10%)15 (4.9)8 (53.3)3 (20.0)4 (26.7)
      High (>10%)135 (44.0)93 (68.9)29 (21.5)13 (9.6)
      PR expression299.004
      Negative (0)166 (55.5)80 (48.2)38 (22.9)48 (28.9)
      Low (1-20%)53 (17.7)38 (71.7)8 (15.1)7 (13.2)
      High (>20%)80 (26.8)50 (65.0)18 (22.5)10 (12.5)
      HER2 overexpression/amplification307.854
      (IHC and ISH)
      Present138 (45.0)78 (56.5)32 (23.2)28 (20.3)
      Absent169 (55.0)97 (57.4)35(20.7)37 (21.9)
      Ki67 proliferative index941.000
      Low (1%-15%)7 (7.4)4 (57.1)1 (14.3)2 (28.6)
      High (> 15%)87 (92.6)43 (49.4)19 (21.8)25 (28.7)
      TLS312<.001
      Present37 (11.9)6 (16.2)10 (27.0)21 (56.8)
      Absent275 (88.1)170 (61.8)60 (21.8)45 (16.4)
      Lymphoid aggregate311.004
      Present88 (28.3)49 (55.7)29 (33.0)10 (11.4)
      Absent223 (71.7)127 (57)41 (18.4)55 (24.7)
      Intensity of inflammation in non-244.715
      tumoral breast parenchyma
      Mild (1%-10%)162 (66.4)93 (57.4)37 (22.8)32 (19.8)
      Moderate (11%-49%)50 (20.5)25 (50.0)12 (24.0)13 (26.0)
      High (≥ 50%)32 (13.1)15 (46.9)8 (25.0)9 (28.1)
      Accompanying DCIS312.439
      Present132 (42.3)80 (60.6)27 (20.5)25 (18.9)
      Absent180 (57.7)96 (53.3)43 (23.9)41 (22.8)
      TIL density in accompanying DCIS132<.001
      Mild (1%-10%)77 (58.3)58 (75.3)12 (15.6)7 (9.1)
      Moderate (11%-49%)22 (16.7)11 (50.0)9 (40.9)2 (9.1)
      High (≥50%)33 (25.0)11 (33.3)5 (15.2)17 (51.5)
      Stil = Stromal tumor infiltrating lymphocytes; SD = Standard Deviation; IHC = Immunohistochemistry; ISH = In situ hybridization
      The effect of TIL density on the overall survival of 312 cases diagnosed with invasive breast carcinoma was investigated. In univariate analyzes performed with the Kaplan-Meier method, a statistically significant relationship was determined between stromal TIL density and overall survival only in the HR (-)/HER2 (+) group (P = .007) (Figure 1). Overall survival was higher in cases with high stromal TIL density. In this group, there was no statistically significant relationship between the stromal TIL density and disease-free survival (P = 0.064). Nevertheless, disease-free survival tends to be lower in those cases with lower stromal TIL density. The effect of stromal TIL density on survival was investigated by univariate Cox regression test and a statistically significant association was found between overall survival and TIL (Hazard ratio (HazR) = 0.6, 95% confidence interval (CI) = 0.4-0.9, P = .050), and also between disease-free survival and TIL (HazR = 0.5, 95% CI = 0.3-0.9, P = .042) in invasive breast carcinoma cases. In the multivariate Cox regression test, it was determined that tumor stage, stromal TIL density, ER expression, and HER2 overexpression were each determined to be independent prognostic factors, in terms of overall survival. In addition, the stromal TIL density and ER expression have been shown to be independent prognostic factors in terms of disease-free survival (Table 2).
      Figure 1
      Figure 1Association of overall and disease-free survival with TIL, TLS, and PD-1 in invasive breast tumors.
      Table 2Cox Regression Analysis for Overall Survival and Disease-Free Survival in Invasive Breast Tumors
      UnivariateMultivariate
      Overall Survival n = 312HazR%95 CIPHazR%95 CIP
      Age at diagnosis (≥ 40 / < 40 years)1.0460.572-1.913.883---
      Tumor size (> 2 / ≤ 2 cm)2.3561.260-4.404.0071.8040.938-3.469.077
      Lymph node metastasis (present / absent)2.6721.526-4.679.0011.3310.645-2.747.439
      Stage (III-IV / I-II)3.7202.280-6.069<.0013.5131.832-6.736<.001
      Density of stromal TIL (moderate-high / mild)0.6020.363-0.999.0500.4530.260-0.791.005
      TLS (present / absent)0.2260.055-0.923.0380.1450.020-1.064.058
      ER expression (positive / negative)0.5320.327-0.864.0110.3540.210-0.599<.001
      HER2 over expression (present/ absent)0.6200.381-1.010.0550.4470.265-0.754.003
      Disease-free survival n = 169
      Age at diagnosis (≥ 40 / < 40 years)0.7070.366-1.367.303---
      Tumor size (> 2 / ≤ 2 cm)1.7470.863-3.537.1211.4660.691-3.113.319
      Lymph node metastasis (present / absent)2.2171.145-4.293.0181.7600.771-4.017.179
      Stage (III-IV / I-II)0.4930.274-0.887.0181.6110.764-3.400.210
      Density of stromal TIL (moderate-high / mild)0.5050.262-0.976.0420.3920.189-0.813.012
      TLS (present / absent)0.1870.026-1.356.0970.2630.035-1.985.195
      ER expression (positive / negative)0.7200.400-1.298.2750.5240.276-0.996.049
      HER2 over expression (present/ absent)1.0410.583-1.858.8930.7390.382-1.429.368

      TLS: Relationship with Pathological Features and Prognosis

      A statistically significant relationship was observed between TLS and neoadjuvant treatment, relapse and death (P= .025, P= .043, and P= .009, respectively). More TLS were observed in basal-like phenotype and medullary-like invasive ductal carcinomas (P= .048), as in triple (-) and HR (-)/HER2 (+) tumors (P= .019). ER and PR negativity were found to be associated with the presence of TLS (P= .004 and p = 0.013, respectively). A higher rate of TLS were observed in tumors with higher stromal TIL density (P< .001) (Supplementary Table 2).
      In univariate analyzes performed with the Kaplan-Meier method, a statistically significant relationship was found between the presence of TLS and overall survival (P= .023) (Figure 1). Overall survival was higher with TLS, and there was no statistically significant relationship between the presence of TLS and disease-free survival (P= .062). However, it has been observed that patients with TLS tend to have higher disease-free survival. Overall survival analysis performed with univariate Cox regression test revealed higher overall survival in the presence of of TLS (HazR = 0.2, 95% CI 0.1-0.9, P= .038). No independent prognostic factor could be shown in multivariate analyzes (Table 2).

      PD-1, LAG-3, and TIM-3 Expressions: Relationship With Pathological Features and Prognosis

      Higher PD-1 expression was observed on intratumoral TIL in cases who did not receive neoadjuvant therapy, did not relapse, and did not die (P= .012, P= .001, and P< .001, respectively), as well as in cases who had higher stromal TIL density and had TLS (P< .001 and P= .001, respectively). However, no statistically significant relationship was found between other histopathological features and PD-1 expression on TIL. PD-1 expression on the stromal TIL was observed to be higher in cases having invasive ductal carcinomas in the medullary pattern, HR (-)/HER2 (+) immunoprofile, and high stromal TIL density (P= .037, P= .001, and P= .002, respectively). High stromal PD-1 expression was found to be statistically significant in HER2 (+) cases when analyzed considering only HER2 expression, regardless of HR status (P< .001) (Table 3).
      Table 3The Relationship Between PD-1 Expression on iTIL and sTIL With Clinicopathological and Histological Parameters in Invasive Breast Tumors
      PD-1 Expression on iTIL, n (%)PD-1 Expression on sTIL, n (%)
      n (%)Low (< 5%)High (≥ 5%)PLow (<5%)High (≥5%)P
      Age at diagnosis99.056.040
      < 4016 (16.2)7 (43.8)9 (56.3)4 (25.0)12 (75.0)
      ≥ 4083 (83.8)57 (68.7)26 (31.3)44 (53.0)39 (47.0)
      pT97.536.084
      pT128 (28.9)17 (60.7)11 (39.3)17 (60.7)11 (39.3)
      pT258 (59.8)40 (69)18 (31)29 (50.0)29 (50.0)
      pT310 (10.3)5 (50.0)5 (50.0)2 (20.0)8 (80.0)
      pT41 (1.0)1 (100)0 (-)0 (-)1 (100)
      pN98.130.092
      pN039 (39.8)27 (69.2)12 (30.8)24 (61.3)15 (38.5)
      pN123 (23.5)14 (60.9)9 (39.1)10 (43.5)13 (56.5)
      pN216 (16.3)7 (43.8)9 (56.3)4 (25.0)12 (75.0)
      pN320 (20.4)16 (80.0)4 (20.0)10 (50.0)10 (50.0)
      pM981.000.490
      pM097 (99)63 (64.9)34 (35.1)47 (48.5)50 (51.5)
      pM11 (1.0)1 (100)0 (-)1 (100)0 (-)
      TNM stages99.932.055
      I22 (22.4)15 (68.2)7 (31.8)15 (68.2)7 (31.8)
      II36 (36.7)24 (66.7)12 (33.3)18 (50.0)18 (50.0)
      III39 (39.8)24 (61.5)15 (38.5)14 (35.9)25 (64.1)
      IV1 (1.0)1 (100)0 (-)1 (100)0 (-)
      Neoadjuvant therapy99.012.878
      Present15 (15.2)14 (93.3)1 (6.7)7 (46.7)8 (53.3)
      Absent84 (84.8)50 (59.5)34 (40.5)41 (48.8)43 (51.2)
      Death99<.001.384
      Present25 (25.3)24 (96.0)1 (4.0)14 (56.0)11 (44.0)
      Absent74 (74.7)40 (54.1)34 (45.9)34 (45.9)40 (54.1)
      Recurrence99.001.872
      Present22 (22.2)21 (95.5)1 (4.5)11 (50.0)11 (50.0)
      Absent77 (77.8)43 (55.8)34 (44.2)37 (48.1)40 (51.9)
      Histologic type99.135.037
      Invasive ductal carcinoma, NOS77 (77.8)52 (67.5)25 (32.5)35 (45.5)42 (54.5)
      Invasive ductal carcinoma with basal-like phenotype16 (16.2)10 (62.5)6 (37.5)12 (75.0)4 (25.0)
      Invasive ductal carcinoma with medullary pattern5 (5.1)1 (20.0)4 (80.0)1 (20)4 (80)
      Invasive ductal carcinoma with metaplastic features1 (1.0)1 (100)0 (-)0 (-)1 (100)
      Receptor expression98.116.001
      Triple (-)51 (52.0)37 (72.5)14 (27.5)34 (66.7)17 (33.3)
      HR (-)/HER2 (+)23 (23.5)11 (47.8)12 (52.2)5 (21.7)18 (78.3)
      HR (+)/HER2 (+)24 (24.5)16 (66.7)8 (33.3)9 (37.5)15 (62.5)
      Histologic grade99.538.742
      12 (2.0)2 (100)0 (-)1 (50.0)1 (50.0)
      29 (9.1)5 (55.6)4 (44.4)3 (33.3)6 (66.7)
      388 (88.9)57 (64.8)31 (35.2)44 (50.0)44 (50.0)
      ER expression99.748.172
      Negative (0)72 (72.7)48 (66.7)24 (33.3)39 (54.2)33 (45.8)
      Low (1-10%)5 (5.1)3 (60.0)2 (40.0)2 (40.0)3 (60.0)
      High (>10%)22 (22.2)13 (59.1)9 (40.9)7 (31.8)15 (68.2)
      PR expression98.811.249
      Negative (0)69 (70.4)45 (65.2)24 (34.8)34 (49.3)35 (50.7)
      Low (1%-20%)15(15.3)9 (60.0)6 (40.0)5 (33.3)10 (66.7)
      High (> 20%)14 (14.3)10 (71.4)4 (28.6)9 (64.3)5 (35.7)
      HER2 overexpression/ amplification (IHC and ISH)98.117<.001
      Present47 (48)27 (57.4)20 (42.6)14 (29.8)33 (70.2)
      Absent51 (52)37 (72.5)14 (27.5)34 (66.7)17 (33.3)
      Ki67 proliferative index39.5141.000
      Low (1%-15%)2 (5.1)2 (100)0 (-)1 (50.0)1 (50.0)
      High (> 15%)37 (94.9)22 (59.5)15 (40.5)22 (59.5)15 (40.5)
      Density of sTIL99<.001.002
      Mild (1%-10%)44 (44.4)38 (86.4)6 (13.6)29 (65.9)15 (34.1)
      Moderate (11%-49%)29 (29.3)20 (69.0)9 (31.0)13 (44.8)16 (55.2)
      High (≥ 50%)26 (26.3)6 (23.1)20 (76.9)6 (23.1)20 (76.9)
      TLS99.001.108
      Present14 (14.1)3 (21.4)11 (78.6)4 (28.6)10 (71.4)
      Absent85 (85.9)61 (71.8)24 (28.2)44 (51.8)41 (48.2)
      Lymphoid aggregate99.099.283
      Present30 (30.3)23 (76.7)7 (23.3)17 (56.7)13 (43.3)
      Absent69 (69.7)41 (59.4)28 (40.6)31 (44.9)38 (55.1)
      Intensity of inflammation in non-tumoral breast parenchyma75.784.114
      Mild (1%-10%)40 (53.3)23 (57.5)17 (42.5)13 (32.5)27 (67.5)
      Moderate (11%-49%)21 (28.0)11 (52.4)10 (47.6)9 (42.9)12 (57.1)
      High (≥ 50%)14 (18.7)9 (64.3)5 (35.7)9 (64.3)5 (35.7)
      Accompanying DCIS99.123.316
      Present38 (38.4)21 (55.3)17 (44.7)16 (42.1)22 (57.9)
      Absent61 (61.6)43 (70.5)18 (29.5)32 (52.5)29 (47.5)
      TIL density in accompanying DCIS38.105.573
      Mild (1%-10%)16 (42.1)11 (68.8)5 (31.3)8 (50.0)8 (50.0)
      Moderate (11%-49%)9 (23.7)6 (66.7)3 (33.3)4 (44.4)5 (55.6)
      High (≥ 50%)13 (34.2)4 (30.8)9 (69.2)4 (30.8)9 (69.2)
      Itil = Intratumoral tumor infiltrating lymphocytes; sTIL = Stromal tumor infiltrating lymphocytes; IHC = Immunohistochemistry; ISH = In situ hybridization
      In invasive ductal carcinomas with basal-like phenotype and medullary pattern, higher LAG-3 expression was observed in both intratumoral and stromal TIL (P= .001 and P= .015, respectively). High LAG-3 expression in intratumoral TIL was observed in cases with high density of stromal TIL and with TLS (P< .001 and P= .002, respectively), as well as high in stromal TIL (P< .001 and P= .035) (Table 4).
      Table 4The Relationship Between LAG-3 Expression on iTIL and sTIL With Clinicopathological and Histological Parameters in Invasive Breast Tumors
      ParametersPatientsLAG-3 Expression on iTIL, n (%)LAG-3 Expression on sTIL, n (%)
      n (%)Low (<5%)High (≥ 5%)PLow (<5%)High (≥ 5%)P
      Age at diagnosis99.129.383
      < 4016 (16.2)9 (56.3)7 (43.8)6 (37.5)10 (62.5)
      ≥ 4083 (83.8)63 (75.9)20 (24.1)41 (49.4)42 (50.6)
      pT97.115.140
      pT128 (28.9)21 (75.0)7 (25.0)16 (57.1)12 (42.9)
      pT258 (59.8)44 (75.9)14 (24.1)27 (46.6)31 (53.4)
      pT310 (10.3)4 (40.0)6 (60.0)2 (20.0)8 (80.0)
      pT41 (1.0)1 (100)0 (-)0 (-)1 (100)
      pN98.442.145
      pN039 (39.8)31 (79.5)8 (20.5)21 (53.8)18 (46.2)
      pN123 (23.5)14 (60.9)9 (39.1)6 (26.1)17 (73.9)
      pN216 (16.3)11 (68.8)5 (31.3)9 (56.3)7 (43.8)
      pN320 (20.4)15 (75.0)5 (25.0)10 (50.0)10 (50.0)
      pM981.0001.000
      pM097 (99.0)70 (72.2)27 (27.8)46 (47.4)51 (52.6)
      pM11 (1.0)1 (100)0 (-)0 (-)1 (100)
      TNM stages99.834.679
      I22 (22.4)17 (77.3)5 (22.7)12 (54.5)10 (45.5)
      II36 (36.7)26 (72.2)10 (27.8)15 (41.7)21 (58.3)
      III39 (39.8)27 (69.2)12 (30.8)19 (48.7)20 (51.3)
      IV1 (1.0)1 (100)0 (-)0 (-)1 (100)
      Neoadjuvant therapy99.062.622
      Present15 (15.2)14 (93.3)1 (6.7)8 (53.3)7 (46.7)
      Absent84 (84.8)58 (69.0)26 (31.0)39 (46.4)45 (53.6)
      Death99.143.323
      Present25 (25.3)21 (84.0)4 (16.0)14 (56.0)11 (44.0)
      Absent74 (74.7)51 (68.9)23 (31.1)33 (44.6)41 (55.4)
      Recurrence99.2780.216
      Present22 (22.2)18 (81.8)4 (18.2)13 (59.1)9 (40.9)
      Absent77 (77.8)54 (70.1)23 (29.9)34 (44.2)43 (55.8)
      Histologic type99.001.015
      Invasive ductal carcinoma, NOS77 (77.8)61 (79.2)16 (20.8)42 (54.5)35 (45.5)
      Invasive ductal carcinoma with basal-like phenotype16 (16.2)11 (68.8)5 (31.3)5 (31.3)11 (68.8)
      Invasive ductal carcinoma with medullary pattern5 (5.1)0 (-)5 (100)0 (-)5 (100)
      Invasive ductal carcinoma with metaplastic features1 (1.0)0 (-)1 (100)0 (-)1 (100)
      Receptor expression98.740.764
      Triple (-)51 (52.0)37 (72.5)14 (27.5)23 (45.1)28 (54.9)
      HR (-)/HER2 (+)23 (23.5)16 (69.6)7 (30.4)11 (47.8)12 (52.2)
      HR (+)/HER2 (+)24 (24.5)19 (79.2)5 (20.8)13 (54.2)11 (45.8)
      Histologic grade99.478.378
      12 (2.0)2 (100)0 (-)2 (100)0 (-)
      29 (9.1)8 (88.9)1 (11.1)5 (55.6)4 (44.4)
      388 (88.9)62 (70.5)26 (29.5)40 (45.5)48 (54.5)
      ER expression99.775.419
      Negative (0)72 (72.7)53 (73.6)19 (26.4)34 (47.2)38 (52.8)
      Low (1%-10%)5 (5.1)3 (60.0)2 (40.0)1 (20.0)4 (80.0)
      High (>10%)22 (22.2)16 (72.7)6 (27.3)12 (54.5)10 (45.5)
      PR expression98.437.378
      Negative (0)69 (70.4)52 (75.4)17 (24.6)32 (46.4)37 (53.6)
      Low (1%-20%)15(15.3)9 (60.0)6 (40.0)6 (40.0)9 (60.0)
      High (> 20%)14 (14.3)11 (78.6)3 (21.4)9 (64.3)5 (35.7)
      HER2 overexpression/ amplification (IHC and ISH)98.830.555
      Present47 (48.0)35 (74.5)12 (25.5)24 (51.1)23 (48.9)
      Absent51 (52.0)37 (72.5)14 (27.5)23 (45.1)28 (54.9)
      Ki67 proliferative index391.0001.000
      Low (1%-15%)2 (5.1)1 (50.0)1 (50.0)1 (50.0)1 (50.0)
      High (> 15%)37 (94.9)24 (64.9)13 (35.1)13 (35.1)24 (64.9)
      Density of sTIL99<.001<.001
      Mild (1%-10%)44 (44.4)42 (95.5)2 (4.5)31 (70.5)13 (29.5)
      Moderate (11%-49%)29 (29.3)23 (79.3)6 (20.7)13 (44.8)16 (55.2)
      High (≥ 50%)26 (26.3)7 (26.9)19 (73.1)3 (11.5)23 (88.5)
      TLS99.002.035
      Present14 (14.1)5 (35.7)9 (64.3)3 (21.4)11 (78.6)
      Absent85 (85.9)67 (78.8)18 (21.2)44 (51.8)41 (48.2)
      Lymphoid aggregate99.284.586
      Present30 (30.3)24 (80.0)6 (20.0)13 (43.3)17 (56.7)
      Absent69 (69.7)48 (69.6)21 (30.4)34 (49.3)35 850.7)
      Intensity of inflammation in non-tumoral breast parenchyma75.671.577
      Mild (1%-10%)40 (53.3)30 (75.0)10 (25.0)22 (55.0)18 (45.0)
      Moderate (11%-49%)21 (28.0)14 (66.7)7 (33.3)9 (42.9)12 (57.1)
      High (≥ 50%)14 (18.7)9 (64.3)5 (35.7)6 (42.9)8 (57.1)
      Accompanying DCIS99.448.667
      Present38 (38.4)26 (68.4)12 (31.6)17 (44.7)21 (55.3)
      Absent61 (61.6)46 (75.4)15 (24.6)30 (49.2)31 (50.8)
      TIL density in accompanying DCIS38.072.117
      Mild (1%-10%)16 (42.1)14 (87.5)2 (12.5)10 (62.5)6 (37.5)
      Moderate (11%-49%)9 (23.7)6 (66.7)3 (33.3)4 (44.4)5 (55.6)
      High (≥ 50%)13 (34.2)6 (46.2)7 (53.8)3 (23.1)10 (76.9)
      Itil = Intratumoral tumor infiltrating lymphocytes; sTIL = Stromal tumor infiltrating lymphocytes; IHC = Immunohistochemistry; ISH = In situ hybridization
      Considering TIM-3, it was seen that its expression was high on both intratumoral and stromal TIL in invasive ductal carcinomas with basal-like phenotype and medullary pattern (P= .001 and P= .007, respectively). In addition, in cases with high stromal TIL density and with TLS, higher TIM-3 expression was observed on intratumoral TIL (P< .001 and P< .001, respectively), as well as on stromal TIL (P< .001 and P= .013, respectively) (Table 5).
      Table 5The Relationship Between TIM-3 Expression on iTIL and sTIL With Clinicopathological and Histological Parameters in Invasive Breast Tumors
      ParametersPatientsTIM-3 Expression on iTIL n (%)TIM-3 Expression on sTIL n (%)
      n (%)Low (< 5%)High (≥ 5%)PLow (< 5%)High (≥ 5%)P
      Age at diagnosis99.2341.000
      < 4016 (16.2)12 (75.0)4 (25.0)15 (93.8)1 (6.3)
      ≥ 4083 (83.8)73 (88.0)10 (12.0)76 (91.6)7 (8.4)
      pT97.7301.000
      pT128 (28.9)25 (89.3)3 (10.7)26 (92.9)2 (7.1)
      pT258 (59.8)49 (84.3)9 (15.5)53 (91.4)5 (8.6)
      pT310 (10.3)8 (80.0)2 (20.0)9 (90.0)1 (10.0)
      pT41 (1.0)1 (100)0 (-)1 (100)0 (-)
      pN98.580.439
      pN039 (39.8)32 (82.1)7 (17.9)35 (89.7)4 (10.3)
      pN123 (23.5)19 (82.6)4 (17.4)20 (87.0)3 (13.0)
      pN216 (16.3)14 (87.5)2 (12.5)15 (93.8)1 (6.3)
      pN320 (20.4)19 (95.0)1 (5.0)20 (100)0 (-)
      pM981.0001.000
      pM097 (99.0)83 (85.6)14 (14.4)89 (91.8)8 (8.2)
      pM11 (1.0)1 (100)0 (-)1 (100)0 (-)
      TNM stages99.597.665
      I22 (22.4)19 (86.4)3 (13.6)20 (90.9)2 (9.1)
      II36 (36.7)29 (80.6)7 (19.4)32 (88.9)4 (11.1)
      III39 (39.8)35 (89.7)4 (10.3)37 (94.9)2 (5.1)
      IV1 (1.0)1 (100)0 (-)1 (100)0 (-)
      Neoadjuvant therapy99.119.603
      Present15 (15.2)15 (100)0 (-)15 (100)0 (-)
      Absent84 (84.8)70 (83.3)14 (16.7)76 (90.5)8 (9.5)
      Death99.110.675
      Present25 (25.3)24 (96.0)1 (4.0)24 (96.0)1 (4.0)
      Absent74 (74.7)61(82.4)13 (17.6)67 (90.5)7 (9.5)
      Recurrence99.183.680
      Present22 (22.2)21(95.5)1 (4.5)21 (95.5)1 (4.5)
      Absent77 (77.8)64 (83.1)13 (16.9)70 (90.9)7 (9.1)
      Histologic type99.001.007
      Invasive ductal carcinoma, NOS77 (77.8)71 (92.2)6 (7.8)74 (96.1)3 (3.9)
      Invasive ductal carcinoma with basal- like phenotype16 (16.2)12 (75.0)4 (25.0)13 (81.3)3 (18.8)
      Invasive ductal carcinoma with medullary pattern5 (5.1)2 (40.0)3 (60.0)4 (80.0)1 (20.0)
      Invasive ductal carcinoma with metaplastic features1 (1.0)0 (-)1 (100)0 (-)1 (100)
      Receptor expression98.065.89
      Triple (-)51 (52.0)42 (82.4)9 (17.6)44 (86.5)7 (13.7)
      HR (-)/HER2 (+)23 (23.5)19 (82.6)4 (17.4)22 (95.7)1 (4.3)
      HR (+)/HER2 (+)24 (24.5)24 (100)0 (-)24 (100)0 (-)
      Histologic grade99.5271.000
      12 (2.0)2 (100)0 (-)2 (100)0 (-)
      29 (9.1)9 (100)0 (-)9 (100)0 (-)
      388 (88.9)74 (84.1)14 (15.9)80 (90.9)8 (9.1)
      ER expression99.565.281
      Negative (0)72 (72.7)61 (84.7)11 (15.3)64 (88.9)8 (11.1)
      Low (1%-10%)5 (5.1)4 (80.0)1 (20.0)5 (100)0 (-)
      High (> 10%)22 (22.2)20 (90.9)2 (9.1)22 (100)0 (-)
      PR expression98.397.297
      Negative (0)69 (70.4)58 (84.1)11 (15.9)61 (88.4)8 (11.6)
      Low (1%-20%)15(15.3)13 (86.7)2 (13.3)15 (100)0 (-)
      High (> 20%)14 (14.3)14 (100)0 (-)14 (100)0 (-)
      HER2 overexpression/ amplification (IHC and ISH)98.183.061
      Present47 (48.0)43 (91.5)4 (8.5)46 (97.9)1 (2.1)
      Absent51 (52.0)42 (82.4)9 (17.6)44 (86.3)7 (13.7)
      Ki67 proliferative index39.413.287
      Low (1%-15%)2 (5.1)1 (50.0)1 (50.0)1 (50.0)1 (50.0)
      High (> 15%)37 (94.9)29 (78.4)8 (21.6)32 (86.5)5 (13.5)
      Density of stromal TIL99<.001<.001
      Mild (1%-10%)44 (44.4)44 (100)0 (-)44 (100)0 (-)
      Moderate (11%-49%)29 (29.3)28 (96.6)1 (3.4)29 (100)0 (-)
      High (≥ 50%)26 (26.3)13 (50.0)13 (50.0)18 (69.2)8 (30.8)
      TLS99<.001.013
      Present14 (14.1)7 (50.0)7 (50.0)10 (71.4)4 (28.6)
      Absent85 (85.9)78 (91.8)7 (8.2)81 (95.3)4 (4.7)
      Lymphoid aggregate99.058.429
      Present30 (30.3)29 (96.7)1 (3.3)29 (96.7)1 (3.3)
      Absent69 (69.7)56 (81.2)13 (18.8)62 (89.9)7 (10.1)
      Intensity of inflammation in non-tumoral breast parenchyma75.355.038
      Mild (1%-10%)40 (53.3)35 (87.5)5 (12.5)38 (95.0)2 (5.0)
      Moderate (11%-49%)21 (28.0)17 (81.0)4 (19.0)20 (95.2)1 (4.8)
      High (≥ 50%)14 (18.7)10 (71.4)4 (28.6)10 (71.4)4 (28.6)
      Accompanying DCIS99.8251.000
      Present38 (38.4)33 (86.8)5 (13.2)35 (92.1)3 (7.9)
      Absent61 (61.6)52 (85.2)9 (14.8)56 (91.8)5 (8.2)
      TIL density in accompanying DCIS38.415.310
      Mild (1%-10%)16 (42.1)15 (93.8)1 (6.3)16 (100)0 (-)
      Moderate (11%-49%)9 (23.7)8 (88.9)1 (11.1)8 (88.9)1 (11.1)
      High (≥ 50%)13 (34.2)10 (76.9)3 (23.1)11 (84.6)2 (15.4)
      Itil = Intratumoral tumor infiltrating lymphocytes; sTIL = Stromal tumor infiltrating lymphocytes; IHC = Immunohistochemistry; ISH = In situ hybridization
      When DCIS cases were evaluated, no significant difference was found between PD-1 and/or LAG-3 expressions on intratumoral and/or stromal TIL and clinicopathological parameters. By the way, TIM-3 expression was not detected on intratumoral TIL at all. High TIM-3 expression was observed on stromal TIL in those cases with moderate to high stromal TIL density and with TLS (P= .050 and P= .009, respectively).
      In univariate analyzes performed with the Kaplan-Meier method, a statistically significant relationship was found between PD-1 expression in the intratumoral TIL and overall survival and disease-free survival (P< .001 and P= .001, respectively) (Figure 1). Overall survival and disease-free survival also tend to be higher in cases with high PD-1 expression on intratumoral TIL. However, no significant relationship was found between survival and other marker expressions on TIL (ie, PD-1 expression on stromal TIL, LAG-3 and TIM-3 expression on both intratumoral and stromal TIL). Nevertheless, patients with higher expression of these receptors tend to have higher overall survival and disease-free survival rates. These results were evaluated with the univariate Cox regression test. Among these 3 biomarkers, a statistically significant difference was found only in relation to PD-1. This difference appeared as higher PD-1 expression on intratumoral TIL associates with higher overall (HazR = 0.07, 95% CI 0.009-0.504, P= .009) and also disease-free survival rates (HazR = 0.07, 95% CI 0.01-0.5, P= .011). In the multivariate Cox regression test which was performed to investigate PD-1 expression on intratumoral TIL in terms of independent positive prognostic factor, a statistically significant difference was found between PD-1 expression on intratumoral TIL in terms of overall survival (HazR = 0.05, 95% CI 0.006-0.4, P= .005), as well as disease-free survival (HazR = 0.06, 95% CI 0.007- 0.5, P= .010).

      Discussion

      In terms of high-grade breast carcinomas which this study was conducted, HR (-)/HER2 (+) and triple (-) invasive ductal carcinomas, as well as those showing basal-like phenotype, were shown to have higher stromal TIL density. In comparison, HR (+) breast carcinomas were strikingly found to have lower TIL density, regardless of HER2 status. Similarly, studies in the literature report that invasive ductal carcinomas with basal-like phenotype, triple (-), and HER2 (+) tumors have higher TIL density.
      • Denkert C
      • von Minckwitz G
      • Darb-Esfahani S
      • et al.
      Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.
      • Loi S
      • Michiels S
      • Salgado R
      • et al.
      Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial.
      • Kim S
      • Park S
      • Cho MS
      • Lim W
      • Moon BI
      • Sung SH.
      Strong correlation of indoleamine 2,3-dioxygenase 1 expression with basal-like phenotype and increased lymphocytic infiltration in triple-negative breast cancer.
      • Heng YJ
      • Lester SC
      • Tse GM
      • et al.
      The molecular basis of breast cancer pathological phenotypes.
      It has also been reported that the basal-like phenotype that occurs in breast carcinomas is associated with increased mutation burden. Likewise, triple (-) and HER2 (+) groups were found to have a higher tumor mutation burden compared to luminal groups.
      • Thomas A
      • Routh ED
      • Pullikuth A
      • et al.
      Tumor mutational burden is a determinant of immune-mediated survival in breast cancer.
      It is known that more inflammatory cell infiltration occurs in the tumor microenvironment due to increased neoantigens in tumors with increased mutation burden.
      • Abbas AK
      • Lichtman AH
      • Pillai S.
      Cellular and Molecular Immunology E-Book.
      It has also been shown that there is more pro-inflammatory cytokine [tumor necrosis factor-alpha (TNF-α) and interleukin-beta (IL-β)] release in basal-like phenotype breast cancers than in luminal A cancers.
      • Liubomirski Y
      • Lerrer S
      • Meshel T
      • et al.
      Tumor-stroma-inflammation networks promote pro-metastatic chemokines and aggressiveness characteristics in triple-negative breast cancer.
      These data explain the high TIL density in cases diagnosed with basal-like invasive ductal carcinoma and in HER2 (+) cases.
      In the classification made considering only HER2, no significant relationship was found between stromal TIL density and HER2 expression. In some studies, it is reported that the molecular intrinsic subtype with the highest mutation burden is the HER2 dominant subtype.
      • Thomas A
      • Routh ED
      • Pullikuth A
      • et al.
      Tumor mutational burden is a determinant of immune-mediated survival in breast cancer.
      However, this molecular subtype is a heterogeneous group in terms of immunohistochemical expressions of ER, PR, and HER2, and it is difficult to determine this molecular subtype by taking into account only the HER2 expression.
      • Prat A
      • Pineda E
      • Adamo B
      • et al.
      Clinical implications of the intrinsic molecular subtypes of breast cancer.
      ,
      • Jamshidi N
      • Yamamoto S
      • Gornbein J
      • Kuo MD.
      Receptor-based surrogate subtypes and discrepancies with breast cancer intrinsic subtypes: implications for image biomarker development.
      To give an example, in a gene expression analysis study including PAM50, Prat et al. found that %44 of HER2 (+) tumors were immunohistochemically compatible with HER2 dominant, %26.8 luminal B, 17.6% luminal A, and 11% basal-like subtypes.
      • Prat A
      • Pineda E
      • Adamo B
      • et al.
      Clinical implications of the intrinsic molecular subtypes of breast cancer.
      This heterogeneity may be the reason why no significant relationship could be shown in our study in terms of TIL density when only classification by HER2 expression considered.
      There are many studies in the literature that have investigated the relationship between TIL and survival in breast cancer population with different clinical characteristics.
      • Denkert C
      • von Minckwitz G
      • Darb-Esfahani S
      • et al.
      Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.
      ,
      • Loi S
      • Michiels S
      • Salgado R
      • et al.
      Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial.
      ,
      • Loi S
      • Sirtaine N
      • Piette F
      • et al.
      Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98.
      • Salgado R
      • Denkert C
      • Campbell C
      • et al.
      Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab: a secondary analysis of the neoaltto trial.
      • Liu JY
      • Yang GF
      • Chen FF
      • Peng CW.
      Evaluating the prognostic significance of tumor-infiltrating lymphocytes in solid tumor: practice of a standardized method from the international immuno-oncology biomarkers working group.
      • Jang N
      • Kwon HJ
      • Park MH
      • Kang SH
      • Bae YK.
      Prognostic value of tumor-infiltrating lymphocyte density assessed using a standardized method based on molecular subtypes and adjuvant chemotherapy in invasive breast cancer.
      Of these studies, Denkert et al. had the largest number of cases with 3771 breast cancer cases that received neoadjuvant therapy in 2018. In this study, which evaluated core biopsies before neoadjuvant therapy, it was found that overall survival also increased with an increase in TIL density in triple (-) breast cancers, while no significant association was found in HER2 (+) breast tumors. However, overall survival has been reported to decrease with an increase in TIL in luminal HER2 (-) breast tumors. It was also mentioned that disease-free survival in triple (-) breast tumors and HER2 (+) breast tumors increased with the increase of TIL, and no significant relationship was found in this regard in luminal HER2 (-) breast tumors
      • Denkert C
      • von Minckwitz G
      • Darb-Esfahani S
      • et al.
      Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.
      In our study, although no significant difference was found in terms of overall survival and disease-free survival with the groups determined as mild-moderate-high stromal TIL regarding all cases, it was found that the group with mild TIL had lower overall and disease-free survival. In the analyzes performed according to the receptor expressions, unlike the above-mentioned study of Denkert et al. a significant relationship was found between the overall survival in the HR (-)/HER2 (+) group and the stromal TIL density rates, and it was determined that those patients with high TIL density had longer overall survival. In multivariate survival analyzes performed by combining moderate and high TIL density groups (as moderate/high vs. mild), stromal TIL density was found to be an independent prognostic factor in terms of overall survival and disease-free survival. This result was interpreted as “high stromal TIL density in high-grade breast carcinomas is an indicator of good prognosis.”
      In addition to TIL, another subject that has been investigated in recent years, is the relation of TLS, which are ectopic lymphoid formations that may accompany the tumor and resemble the secondary lymphoid organs, with prognosis.
      • Hiraoka N
      • Ino Y
      Yamazaki-Itoh R. tertiary lymphoid organs in cancer tissues.
      In many studies, the presence of TLS formation in different cancers (lung cancer, colon cancer, and melanoma) has been investigated in terms of prognosis.
      • Dieu-Nosjean MC
      • Antoine M
      • Danel C
      • et al.
      Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures.
      • Trajkovski G
      • Ognjenovic L
      • Karadzov Z
      • et al.
      Tertiary lymphoid structures in colorectal cancers and their prognostic value.
      • Cabrita R
      • Lauss M
      • Sanna A
      • et al.
      Tertiary lymphoid structures improve immunotherapy and survival in melanoma.
      In our study, it was observed that the presence of TLS in breast tumors was more common in those with high TIL density. This finding is also mentioned in several studies in the literature.
      • Liu X
      • Tsang JYS
      • Hlaing T
      • et al.
      Distinct tertiary lymphoid structure associations and their prognostic relevance in HER2 positive and negative breast cancers.
      ,
      • Figenschau SL
      • Fismen S
      • Fenton KA
      • Fenton C
      • Mortensen ES.
      Tertiary lymphoid structures are associated with higher tumor grade in primary operable breast cancer patients.
      • Lee HJ
      • Kim JY
      • Park IA
      • et al.
      Prognostic significance of tumor-infiltrating lymphocytes and the tertiary lymphoid structures in her2-positive breast cancer treated with adjuvant trastuzumab.
      • Lee HJ
      • Park IA
      • Song IH
      • et al.
      Tertiary lymphoid structures: prognostic significance and relationship with tumour-infiltrating lymphocytes in triple-negative breast cancer.
      When we look at the receptor expression status, the TLS were most frequently observed in the HR (-)/HER2 (+) group and followed by in the triple (-) group. We also found a significant association between the presence of TLS and overall survival in univariate analyses determining that overall survival was higher in the presence of TLS. Regarding TLS and survival, different outcomes were mentioned in literature. Such that, in 2 studies, when all molecular intrinsic subtypes of breast carcinomas were evaluated together, no relationship was found between TLS and survival
      • Liu X
      • Tsang JYS
      • Hlaing T
      • et al.
      Distinct tertiary lymphoid structure associations and their prognostic relevance in HER2 positive and negative breast cancers.
      ,
      • Lee HJ
      • Kim JY
      • Park IA
      • et al.
      Prognostic significance of tumor-infiltrating lymphocytes and the tertiary lymphoid structures in her2-positive breast cancer treated with adjuvant trastuzumab.
      ; but, when Liu et al. evaluated HER2 (+) breast tumors separately, they found that TLS was associated with longer disease-free survival.
      • Liu X
      • Tsang JYS
      • Hlaing T
      • et al.
      Distinct tertiary lymphoid structure associations and their prognostic relevance in HER2 positive and negative breast cancers.
      PD-1, LAG-3, and TIM-3, three less investigated immune checkpoint molecules which our study focuses on, are targets that may be promising in immunotherapy. In our study, we examined the relationship of these receptors with prognostic parameters and also with survival in breast cancer patients with high tumor grade. We concluded that all these immune checkpoint inhibitor receptors are highly expressed in tumors with high stromal TIL density and in tumors containing TLS.
      Considering PD-1 expression, higher PD-1 expression was found in HER2 (+) tumors, regardless of HR status. This result was found different from some studies in the literature which have reported that stromal and intratumoral high PD-1 expression is more common in triple (-) and basal-like invasive breast tumors.
      • Tsang JY
      • Au WL
      • Lo KY
      • et al.
      PD-L1 expression and tumor infiltrating PD-1+ lymphocytes associated with outcome in HER2+ breast cancer patients.
      • Kitano A
      • Ono M
      • Yoshida M
      • et al.
      Tumour-infiltrating lymphocytes are correlated with higher expression levels of PD-1 and PD-L1 in early breast cancer.
      • Noske A
      • Möbus V
      • Weber K
      Relevance of tumour-infiltrating lymphocytes, PD-1 and PD-L1 in patients with high-risk, nodal-metastasised breast cancer of the German Adjuvant Intergroup Node-positive study.
      • Muenst S
      • Soysal SD
      • Gao F
      • Obermann EC
      • Oertli D
      • Gillanders WE.
      The presence of programmed death 1 (PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer.
      We also found that high PD-1 expression in intratumoral TIL was associated with increased overall and disease-free survival. In multivariate analyzes, it was determined that PD-1 expression in intratumoral TIL was an independent positive prognostic factor in terms of overall and disease-free survival. It was also noted that some studies suggested that there was no significant relationship between PD-1 expression and overall survival and disease-free survival.
      • Tsang JY
      • Au WL
      • Lo KY
      • et al.
      PD-L1 expression and tumor infiltrating PD-1+ lymphocytes associated with outcome in HER2+ breast cancer patients.
      ,
      • Kitano A
      • Ono M
      • Yoshida M
      • et al.
      Tumour-infiltrating lymphocytes are correlated with higher expression levels of PD-1 and PD-L1 in early breast cancer.
      ,
      • Zhou T
      • Xu D
      • Tang B
      • et al.
      Expression of programmed death ligand-1 and programmed death-1 in samples of invasive ductal carcinoma of the breast and its correlation with prognosis.
      ,
      • Bottai G
      • Raschioni C
      • Losurdo A
      • et al.
      An immune stratification reveals a subset of PD-1/LAG-3 double-positive triple-negative breast cancers.
      On the other hand, Yeong et al. reported that increased PD-1 expression was associated with increased overall survival and disease-free survival, in their study including 269 triple (-) breast carcinoma cases.
      • Yeong J
      • Lim JCT
      • Lee B
      • et al.
      Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer.
      In contrast to this study and to ours, there are also several studies in the literature reporting that PD-1 expression is associated with decreased overall and disease-free survival.
      • Muenst S
      • Soysal SD
      • Gao F
      • Obermann EC
      • Oertli D
      • Gillanders WE.
      The presence of programmed death 1 (PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer.
      ,
      • Sun S
      • Fei X
      • Mao Y
      • et al.
      PD-1(+) immune cell infiltration inversely correlates with survival of operable breast cancer patients.
      In our study, high LAG-3 expression on intratumoral and stromal TIL was tended to be higher in patients in terms of survival, but no statistically significant relationship could be shown.
      There are only a few studies with different results in the literature investigating the relationship between survival and LAG-3 expression on TIL in breast cancer. In a study conducted by Bottai et al. concerning 259 triple (-) breast tumors, no statistically significant relationship was found between LAG-3 and survival.
      • Bottai G
      • Raschioni C
      • Losurdo A
      • et al.
      An immune stratification reveals a subset of PD-1/LAG-3 double-positive triple-negative breast cancers.
      Tu et al. found that LAG-3 expression improves post-progression survival in triple (-) breast tumors and disease-free survival in those with a p53 mutation.
      • Tu L
      • Guan R
      • Yang H
      • et al.
      Assessment of the expression of the immune checkpoint molecules PD-1, CTLA4, TIM-3 and LAG-3 across different cancers in relation to treatment response, tumor-infiltrating immune cells and survival.
      In their study, Wang et al. were reported that high LAG-3 expression after neoadjuvant treatment in triple (-) breast tumors is associated with poor prognosis.
      • Wang Y
      • Dong T
      • Xuan Q
      • Zhao H
      • Qin L
      • Zhang Q.
      Lymphocyte-activation gene-3 expression and prognostic value in neoadjuvant-treated triple-negative breast cancer.
      In an animal model with mice evaluating treatment results, co-inhibition of PD-1 and LAG-3 was found to be correlated with a better response to the tumor in triple (-) breast tumors.
      • Du H
      • Yi Z
      • Wang L
      • Li Z
      • Niu B
      • Ren G.
      The co-expression characteristics of LAG3 and PD-1 on the T cells of patients with breast cancer reveal a new therapeutic strategy.
      On the other hand, in a clinical study, Brignone et al. were reported that combined use of paclitaxel and IMP321 (LAG-3 antagonist) in metastatic breast cancer has provided an objective tumor response of 50%.
      • Brignone C
      • Gutierrez M
      • Mefti F
      • et al.
      First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity.
      LAG-3 is thought to be a strong immunotherapy target, although the relationship between LAG-3 expression and survival on TIL has not yet been definitively explained.
      Similar to LAG-3, high TIM-3 expression on intratumoral and stromal TIL was tended to be higher in patients in terms of survival, but no statistically significant correlation could be shown. When looking at the small number of similar studies available in the literature, it was seen that similar results were reported. In the study of Burugu et al. including 3992 breast cancer cases, TIM-3 expression on intratumoral TIL was found to be an independent positive prognostic factor.
      • Burugu S
      • Gao D
      • Leung S
      • Chia SK
      • Nielsen TO.
      TIM-3 expression in breast cancer.
      It has also been reported that high TIM-3 expression in triple (-) breast tumors is associated with increased disease-free survival.
      • Tu L
      • Guan R
      • Yang H
      • et al.
      Assessment of the expression of the immune checkpoint molecules PD-1, CTLA4, TIM-3 and LAG-3 across different cancers in relation to treatment response, tumor-infiltrating immune cells and survival.
      ,
      • Byun KD
      • Hwang HJ
      • Park KJ
      • et al.
      T-Cell immunoglobulin mucin 3 expression on tumor infiltrating lymphocytes as a positive prognosticator in triple-negative breast cancer.
      Only 1 study reported that TIM-3 expression in tumor cells may cause tumor progression.
      • Zhang H
      • Xiang R
      • Wu B
      • Li J
      • Luo G.
      T-cell immunoglobulin mucin-3 expression in invasive ductal breast carcinoma: Clinicopathological correlations and association with tumor infiltration by cytotoxic lymphocytes.
      No clinical studies have yet been found in the literature investigating the therapeutic efficacy of TIM-3 antagonists in breast tumors. Similar to LAG-3 and PD-1, TIM-3 may be one of the targets to be used for immunotherapy in the near future.
      Regarding DCIS, which is another important tumor type usually forming a breast lump with indistinct borders, studies by evaluating TIL were also conducted in recent years in order to get some information about the prognosis of those patients.
      • Pruneri G
      • Lazzeroni M
      • Bagnardi V
      • et al.
      The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast.
      • Chen XY
      • Yeong J
      • Thike AA
      • Bay BH
      • Tan PH.
      Prognostic role of immune infiltrates in breast ductal carcinoma in situ.
      • Toss MS
      • Miligy I
      • Al-Kawaz A
      • et al.
      Prognostic significance of tumor-infiltrating lymphocytes in ductal carcinoma in situ of the breast.
      • Thike AA
      • Chen X
      • Koh VCY
      • et al.
      Higher densities of tumour-infiltrating lymphocytes and CD4(+) T cells predict recurrence and progression of ductal carcinoma in situ of the breast.
      In our study, high stromal TIL density rate was found in 8.8% of DCIS, especially in grade 3 tumors; however, such TIL density was not seen in those with microinvasion. Besides, no statistically significant relationship was found between TIL density and microinvasion. In the study of Beguinot et al. in which microinvasive DCIS and pure DCIS were compared, it was found that microinvasive carcinomas have higher TIL density; however, it has been reported that the difference is not statistically significant.
      • Thike AA
      • Chen X
      • Koh VCY
      • et al.
      Higher densities of tumour-infiltrating lymphocytes and CD4(+) T cells predict recurrence and progression of ductal carcinoma in situ of the breast.
      • Beguinot M
      • Dauplat MM
      • Kwiatkowski F
      • et al.
      Analysis of tumour-infiltrating lymphocytes reveals two new biologically different subgroups of breast ductal carcinoma in situ.
      The healing phenomenon was first described in 1934 by Muir et al. as the spontaneous disappearance of DCIS and its replacement by fibrous tissue.
      • Muir R
      • Aitkenhead AC.
      The healing of intra-duct carcinoma of the mamma.
      This condition is characterized by inflammation and fibrosis of the periductal stroma containing the tumor.
      • Rosen PP.
      Rosen's Breast Pathology.
      In our study, a significant relationship was found between the healing phenomenon and TIL density in DCIS. Forty percent of cases with sign of healing phenomenon were included in high TIL density group, while only 2.4% of cases not showing this feature were in this group. Similar results were also noted by Morita et al.
      • Morita M
      • Yamaguchi R
      • Tanaka M
      • et al.
      CD8(+) tumor-infiltrating lymphocytes contribute to spontaneous "healing" in HER2-positive ductal carcinoma in situ.
      In our study, 2 of 68 patients with DCIS died of non-tumor causes and no recurrence was observed during the follow-up period. Therefore, survival analysis could not be performed in the DCIS group. However, in a study including 1488 DCIS cases which was conducted by Pruneri et al., it was showed that 245 patients recurred as in situ or as invasive tumors during follow-up, and there was no significant relationship between stromal TIL and recurrence
      • Pruneri G
      • Lazzeroni M
      • Bagnardi V
      • et al.
      The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast.
      .
      In our study, PD-1 expression on intratumoral TIL was not associated with any of the clinical and histopathological parameters in DCIS. However, PD-1 expression on stromal TIL was found to be related to TIL density determining that tumors with high stromal TIL density show a higher rate of PD-1 expression. Recently, Ubago et al. reported that 31% of HER2 (+) DCIS have PD-1 expression on TIL.
      • Ubago JM
      • Blanco LZ
      • Shen T
      • Siziopikou KP.
      The PD-1/PD-L1 Axis in HER2+ Ductal Carcinoma In Situ (DCIS) of the breast.
      Regarding LAG-3 and TIM-3 expression in intratumoral and stromal TIL, no significant association was found with any of the clinical or histopathological parameters in DCIS. No study has been found on this new subject in the literature yet.

      Conclusion

      As a conclusion, the relationship between TIL, as well as TLS and prognostic factors is an important and significant issue particularly in high grade invasive tumors. Stating the percentage of TIL and the presence of TLS in pathology reports will help to predict the prognosis of the patient and also to contribute the treatment modality. Another important issue we would like to mention is that PD-1 expression on intratumoral TIL is a positive prognostic factor in terms of survival in invasive breast cancer. Other receptor expressions, such as LAG-3 and TIM-3, also may indicate good prognosis; however, further studies on these new markers are needed. Immunotherapy seems to be a promising treatment option for those patients with high grade tumors and whose tumors show marked immune response and express these novel immune checkpoint molecules on TIL.

      Clinical Practice Point

      • Stromal TIL density was found to be an independent prognostic factor in terms of overall survival and disease-free survival in high-grade breast carcinomas.
      • PD-1 expression on intratumoral TIL in invasive breast carcinomas was an independent positive prognostic factor in terms of overall and disease-free survival.
      • High LAG-3 and TIM-3 expression on intratumoral and stromal TIL was tended to be higher in patients in terms of survival.

      Data Availability Statement

      The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

      Ethics Approval

      The study was approved by the Gazi University Faculty of Medicine Clinical Research Ethics Committee in terms of ethics on April 29, 2019, with the meeting number 04.

      Author Contributions

      E.A., G.E. and, A.D. performed study concept and design, development of methodology; E.A, G.E and, O.Y. performed development of writing and review of the paper; E.A. and G.E. provided acquisition, analysis and, interpretation of data, and statistical analysis. All authors read and approved the final paper.

      Acknowledgments

      I would like to thank Gazi University Scientific Research Projects for supporting my project numbered 01/2020-18 .

      Disclosure

      The authors declare that they have no conflict of interest.

      Appendix. Supplementary materials

      References

        • Bray F
        • Ferlay J
        • Soerjomataram I
        • Siegel RL
        • Torre LA
        • Jemal A.
        Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
        CA Cancer J Clin. 2018; 68: 394-424https://doi.org/10.3322/caac.21492
        • Prat A
        • Pineda E
        • Adamo B
        • et al.
        Clinical implications of the intrinsic molecular subtypes of breast cancer.
        Breast. 2015; 24 (Edinburgh, Scotland): S26-S35https://doi.org/10.1016/j.breast.2015.07.008
        • Brignone C
        • Gutierrez M
        • Mefti F
        • et al.
        First-line chemoimmunotherapy in metastatic breast carcinoma: combination of paclitaxel and IMP321 (LAG-3Ig) enhances immune responses and antitumor activity.
        J Transl Med. 2010; 8: 71https://doi.org/10.1186/1479-5876-8-71
      1. Emens LA, Braiteh FS, Cassier P, et al. Inhibition of PD-L1 by MPDL3280A leads to clinical activity in patients with metastatic triple-negative breast cancer (TNBC). AACR; 2015. doi:10.1158/1538-7445.AM2015-2859

        • Migali C
        • Milano M
        • Trapani D
        • et al.
        Strategies to modulate the immune system in breast cancer: checkpoint inhibitors and beyond.
        rapeutic advances med oncol. 2016; 8: 360-374https://doi.org/10.1177/1758834016658423
        • Abbas AK
        • Lichtman AH
        • Pillai S.
        Cellular and Molecular Immunology E-Book.
        Elsevier Health Sciences, California2014
        • Anderson AC
        • Joller N
        • Kuchroo VK.
        Lag-3, Tim-3, and TIGIT: Co-inhibitory receptors with specialized functions in immune regulation.
        Immunity. 2016; 44: 989-1004https://doi.org/10.1016/j.immuni.2016.05.001
        • Dieci MV
        • Radosevic-Robin N
        • Fineberg S
        • et al.
        Update on tumor-infiltrating lymphocytes (TILs) in breast cancer, including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ: A report of the international immuno-oncology biomarker working group on breast cancer.
        Semin Cancer Biol. 2018; 52: 16-25https://doi.org/10.1016/j.semcancer.2017.10.003
        • Stanton SE
        • Disis ML.
        Clinical significance of tumor-infiltrating lymphocytes in breast cancer.
        J Immunother Cancer. 2016; 4: 59https://doi.org/10.1186/s40425-016-0165-6
        • Hiraoka N
        • Ino Y
        Yamazaki-Itoh R. tertiary lymphoid organs in cancer tissues.
        Front Immunol. 2016; 7: 244https://doi.org/10.3389/fimmu.2016.00244
        • Dieu-Nosjean MC
        • Antoine M
        • Danel C
        • et al.
        Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures.
        J Clin Oncol. 2008; 26: 4410-4417https://doi.org/10.1200/JCO.2007.15.0284
        • Trajkovski G
        • Ognjenovic L
        • Karadzov Z
        • et al.
        Tertiary lymphoid structures in colorectal cancers and their prognostic value.
        Open access Macedonian J med sci. 2018; 6: 1824-1828https://doi.org/10.3889/oamjms.2018.341
        • Cabrita R
        • Lauss M
        • Sanna A
        • et al.
        Tertiary lymphoid structures improve immunotherapy and survival in melanoma.
        Nature. 2020; 577: 561-565https://doi.org/10.1038/s41586-019-1914-8
        • Liu X
        • Tsang JYS
        • Hlaing T
        • et al.
        Distinct tertiary lymphoid structure associations and their prognostic relevance in HER2 positive and negative breast cancers.
        Oncologist. 2017; 22: 1316-1324https://doi.org/10.1634/theoncologist.2017-0029
        • Sofopoulos M
        • Fortis SP
        • Vaxevanis CK
        • et al.
        The prognostic significance of peritumoral tertiary lymphoid structures in breast cancer.
        Cancer Immunol Immunother. 2019; 68: 1733-1745https://doi.org/10.1007/s00262-019-02407-8
        • Salgado R
        • Denkert C
        • Demaria S
        • et al.
        The evaluation of tumor-infiltrating lymphocytes (TILs) in breast cancer: recommendations by an International TILs Working Group 2014.
        Ann Oncol. 2015; 26: 259-271https://doi.org/10.1093/annonc/mdu450
        • Denkert C
        • von Minckwitz G
        • Darb-Esfahani S
        • et al.
        Tumour-infiltrating lymphocytes and prognosis in different subtypes of breast cancer: a pooled analysis of 3771 patients treated with neoadjuvant therapy.
        Lancet Oncol. 2018; 19: 40-50https://doi.org/10.1016/S1470-2045(17)30904-X
        • Loi S
        • Michiels S
        • Salgado R
        • et al.
        Tumor infiltrating lymphocytes are prognostic in triple negative breast cancer and predictive for trastuzumab benefit in early breast cancer: results from the FinHER trial.
        Ann Oncol. 2014; 25: 1544-1550https://doi.org/10.1093/annonc/mdu112
        • Kim S
        • Park S
        • Cho MS
        • Lim W
        • Moon BI
        • Sung SH.
        Strong correlation of indoleamine 2,3-dioxygenase 1 expression with basal-like phenotype and increased lymphocytic infiltration in triple-negative breast cancer.
        J Cancer. 2017; 8: 124-130https://doi.org/10.7150/jca.17437
        • Heng YJ
        • Lester SC
        • Tse GM
        • et al.
        The molecular basis of breast cancer pathological phenotypes.
        J Pathol. 2017; 241: 375-391https://doi.org/10.1002/path.4847
        • Thomas A
        • Routh ED
        • Pullikuth A
        • et al.
        Tumor mutational burden is a determinant of immune-mediated survival in breast cancer.
        Oncoimmunology. 2018; 7 (e1490854)https://doi.org/10.1080/2162402X.2018.1490854
        • Liubomirski Y
        • Lerrer S
        • Meshel T
        • et al.
        Tumor-stroma-inflammation networks promote pro-metastatic chemokines and aggressiveness characteristics in triple-negative breast cancer.
        Front Immunol. 2019; 10: 757https://doi.org/10.3389/fimmu.2019.00757
        • Jamshidi N
        • Yamamoto S
        • Gornbein J
        • Kuo MD.
        Receptor-based surrogate subtypes and discrepancies with breast cancer intrinsic subtypes: implications for image biomarker development.
        Radiology. 2018; 289: 210-217https://doi.org/10.1148/radiol.2018171118
        • Loi S
        • Sirtaine N
        • Piette F
        • et al.
        Prognostic and predictive value of tumor-infiltrating lymphocytes in a phase III randomized adjuvant breast cancer trial in node-positive breast cancer comparing the addition of docetaxel to doxorubicin with doxorubicin-based chemotherapy: BIG 02-98.
        J Clin Oncol. 2013; 31: 860-867https://doi.org/10.1200/JCO.2011.41.0902
        • Salgado R
        • Denkert C
        • Campbell C
        • et al.
        Tumor-infiltrating lymphocytes and associations with pathological complete response and event-free survival in HER2-positive early-stage breast cancer treated with lapatinib and trastuzumab: a secondary analysis of the neoaltto trial.
        JAMA Oncol. 2015; 1: 448-454https://doi.org/10.1001/jamaoncol.2015.0830
        • Liu JY
        • Yang GF
        • Chen FF
        • Peng CW.
        Evaluating the prognostic significance of tumor-infiltrating lymphocytes in solid tumor: practice of a standardized method from the international immuno-oncology biomarkers working group.
        Cancer manage res. 2019; 11: 6815-6827https://doi.org/10.2147/CMAR.S201538
        • Jang N
        • Kwon HJ
        • Park MH
        • Kang SH
        • Bae YK.
        Prognostic value of tumor-infiltrating lymphocyte density assessed using a standardized method based on molecular subtypes and adjuvant chemotherapy in invasive breast cancer.
        Ann Surg Oncol. 2018; 25: 937-946https://doi.org/10.1245/s10434-017-6332-2
        • Figenschau SL
        • Fismen S
        • Fenton KA
        • Fenton C
        • Mortensen ES.
        Tertiary lymphoid structures are associated with higher tumor grade in primary operable breast cancer patients.
        BMC Cancer. 2015; 15: 101https://doi.org/10.1186/s12885-015-1116-1
        • Lee HJ
        • Kim JY
        • Park IA
        • et al.
        Prognostic significance of tumor-infiltrating lymphocytes and the tertiary lymphoid structures in her2-positive breast cancer treated with adjuvant trastuzumab.
        Am J Clin Pathol. 2015; 144: 278-288https://doi.org/10.1309/AJCPIXUYDVZ0RZ3G
        • Lee HJ
        • Park IA
        • Song IH
        • et al.
        Tertiary lymphoid structures: prognostic significance and relationship with tumour-infiltrating lymphocytes in triple-negative breast cancer.
        J Clin Pathol. 2016; 69: 422-430https://doi.org/10.1136/jclinpath-2015-203089
        • Tsang JY
        • Au WL
        • Lo KY
        • et al.
        PD-L1 expression and tumor infiltrating PD-1+ lymphocytes associated with outcome in HER2+ breast cancer patients.
        Breast Cancer Res Treat. 2017; 162: 19-30https://doi.org/10.1007/s10549-016-4095-2
        • Kitano A
        • Ono M
        • Yoshida M
        • et al.
        Tumour-infiltrating lymphocytes are correlated with higher expression levels of PD-1 and PD-L1 in early breast cancer.
        ESMO open. 2017; 2e000150https://doi.org/10.1136/esmoopen-2016-000150
        • Noske A
        • Möbus V
        • Weber K
        Relevance of tumour-infiltrating lymphocytes, PD-1 and PD-L1 in patients with high-risk, nodal-metastasised breast cancer of the German Adjuvant Intergroup Node-positive study.
        Eur J Cancer. 2019; 114 (Oxford, England: 1990): 76-88https://doi.org/10.1016/j.ejca.2019.04.010
        • Muenst S
        • Soysal SD
        • Gao F
        • Obermann EC
        • Oertli D
        • Gillanders WE.
        The presence of programmed death 1 (PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer.
        Breast Cancer Res Treat. 2013; 139: 667-676https://doi.org/10.1007/s10549-013-2581-3
        • Zhou T
        • Xu D
        • Tang B
        • et al.
        Expression of programmed death ligand-1 and programmed death-1 in samples of invasive ductal carcinoma of the breast and its correlation with prognosis.
        Anticancer Drugs. 2018; 29: 904-910https://doi.org/10.1097/CAD.0000000000000683
        • Bottai G
        • Raschioni C
        • Losurdo A
        • et al.
        An immune stratification reveals a subset of PD-1/LAG-3 double-positive triple-negative breast cancers.
        Breast Cancer Res. 2016; 18: 121https://doi.org/10.1186/s13058-016-0783-4
        • Yeong J
        • Lim JCT
        • Lee B
        • et al.
        Prognostic value of CD8 + PD-1+ immune infiltrates and PDCD1 gene expression in triple negative breast cancer.
        J Immunother Cancer. 2019; 7: 34https://doi.org/10.1186/s40425-019-0499-y
        • Sun S
        • Fei X
        • Mao Y
        • et al.
        PD-1(+) immune cell infiltration inversely correlates with survival of operable breast cancer patients.
        Cancer Immunol Immunother. 2014; 63: 395-406https://doi.org/10.1007/s00262-014-1519-x
        • Tu L
        • Guan R
        • Yang H
        • et al.
        Assessment of the expression of the immune checkpoint molecules PD-1, CTLA4, TIM-3 and LAG-3 across different cancers in relation to treatment response, tumor-infiltrating immune cells and survival.
        Int J Cancer. 2020; 147: 423-439https://doi.org/10.1002/ijc.32785
        • Wang Y
        • Dong T
        • Xuan Q
        • Zhao H
        • Qin L
        • Zhang Q.
        Lymphocyte-activation gene-3 expression and prognostic value in neoadjuvant-treated triple-negative breast cancer.
        J breast cancer. 2018; 21: 124-133https://doi.org/10.4048/jbc.2018.21.2.124
        • Du H
        • Yi Z
        • Wang L
        • Li Z
        • Niu B
        • Ren G.
        The co-expression characteristics of LAG3 and PD-1 on the T cells of patients with breast cancer reveal a new therapeutic strategy.
        Int Immunopharmacol. 2020; 78106113https://doi.org/10.1016/j.intimp.2019.106113
        • Burugu S
        • Gao D
        • Leung S
        • Chia SK
        • Nielsen TO.
        TIM-3 expression in breast cancer.
        Oncoimmunology. 2018; 7e1502128https://doi.org/10.1080/2162402X.2018.1502128
        • Byun KD
        • Hwang HJ
        • Park KJ
        • et al.
        T-Cell immunoglobulin mucin 3 expression on tumor infiltrating lymphocytes as a positive prognosticator in triple-negative breast cancer.
        J breast cancer. 2018; 21: 406-414https://doi.org/10.4048/jbc.2018.21.e61
        • Zhang H
        • Xiang R
        • Wu B
        • Li J
        • Luo G.
        T-cell immunoglobulin mucin-3 expression in invasive ductal breast carcinoma: Clinicopathological correlations and association with tumor infiltration by cytotoxic lymphocytes.
        Mol Clin Oncol. 2017; 7: 557-563https://doi.org/10.3892/mco.2017.1360
        • Pruneri G
        • Lazzeroni M
        • Bagnardi V
        • et al.
        The prevalence and clinical relevance of tumor-infiltrating lymphocytes (TILs) in ductal carcinoma in situ of the breast.
        Ann Oncol. 2017; 28: 321-328https://doi.org/10.1093/annonc/mdw623
        • Chen XY
        • Yeong J
        • Thike AA
        • Bay BH
        • Tan PH.
        Prognostic role of immune infiltrates in breast ductal carcinoma in situ.
        Breast Cancer Res Treat. 2019; 177: 17-27https://doi.org/10.1007/s10549-019-05272-2
        • Toss MS
        • Miligy I
        • Al-Kawaz A
        • et al.
        Prognostic significance of tumor-infiltrating lymphocytes in ductal carcinoma in situ of the breast.
        Mod Pathol. 2018; 31 (Inc.): 1226-1236https://doi.org/10.1038/s41379-018-0040-8
        • Thike AA
        • Chen X
        • Koh VCY
        • et al.
        Higher densities of tumour-infiltrating lymphocytes and CD4(+) T cells predict recurrence and progression of ductal carcinoma in situ of the breast.
        Histopathology. 2020; 76: 852-864https://doi.org/10.1111/his.14055
        • Beguinot M
        • Dauplat MM
        • Kwiatkowski F
        • et al.
        Analysis of tumour-infiltrating lymphocytes reveals two new biologically different subgroups of breast ductal carcinoma in situ.
        BMC Cancer. 2018; 18: 129https://doi.org/10.1186/s12885-018-4013-6
        • Muir R
        • Aitkenhead AC.
        The healing of intra-duct carcinoma of the mamma.
        J Pathol Bacteriol. 1934; 38: 117-127https://doi.org/10.1002/PATH.1700380202
        • Rosen PP.
        Rosen's Breast Pathology.
        Lippincott Williams & Wilkins, Mexico2001
        • Morita M
        • Yamaguchi R
        • Tanaka M
        • et al.
        CD8(+) tumor-infiltrating lymphocytes contribute to spontaneous "healing" in HER2-positive ductal carcinoma in situ.
        Cancer Med. 2016; 5: 1607-1618https://doi.org/10.1002/cam4.715
        • Ubago JM
        • Blanco LZ
        • Shen T
        • Siziopikou KP.
        The PD-1/PD-L1 Axis in HER2+ Ductal Carcinoma In Situ (DCIS) of the breast.
        Am J Clin Pathol. 2019; 152: 169-176https://doi.org/10.1093/ajcp/aqz020